A technique using fluorescent dyes for characterizing cells by flow cytometry (FCM) has been of considerable utility in immunohematology and cell biology. Another technique known as laser scanning cytometry (LSCM) has been developed to automatically measure laser excited fluorescence at multiple wavelengths and light scatter from cells on stationery slides (as opposed to a flow as in FCM) that have been treated with one or more fluorescent dyes in order to rapidly determine multiple cellular constituents and other features of the cells.
A laser scanning cytometer can use the methods perfected for FCM and has been shown to provide data equivalent to FCM for DNA analysis of aneuploid specimens, for immunophenotyping, and for analysis of cell proliferation and apoptosis. Because it is microscope based and measures cells on a slide and not in a flow chamber (as used in FCM), records position of each cell on the slide, and has higher resolution, it provides a number of benefits that may make it more suitable for pathology laboratories than FCM.
FCM has used fluorescent dyes to quantify cell constituents because fluorescence emissions are directly proportional to the mass of the constituent stained by the dye, if dye concentrations are low. Additionally, light scatter which has been useful to characterize cellular size and granularity occurs at a different wavelength than fluorescence and is easily separated from it in FCM. Automated fluorescence image analysis (FIA) in which a specimen is illuminated by an arc lamp or laser light source and is imaged at one or more wavelengths, using a CCD camera, has also been characterized for use in cell analysis.
The technologies of FIA, FCM and LSCM can be utilized to quantify cell constituents using fluorescence. Two of these, FCM and LSCM measure scatter as well as fluorescence. FIA, FCM, and LSCM each automatically measure fluorescence at multiple wavelengths of cells that have been treated with one or more fluorescent dyes in order to rapidly assay multiple cellular constituents. In FCM and LSCM, fluorescence and scatter result from interaction of the cells with a laser beam comparable in spot size to the cell. The laser optics is designed to produce a large depth of field with nearly collimated excitation to achieve accurate constituent measurements independent of cell position in the FCM stream or LSCM slide focus. In FIA the cells are uniformly illuminated, preferably by a mercury or xenon arc epi-illuminator. Fluorescence is imaged at high resolution and low depth of focus by a sensitive CCD camera. Commercial FIA, FCM and LSCM instruments provide feature values, for each event found, in standardized format computer list mode files.
In LSCM, the cells are measured and retained on a solid support such as a slide. In FCM, cells flow past the laser in a flow of cells which end in a waste container. The LSCM slide position and laser beam are moved under computer control to excite the cells. Since the position of the slide and laser beam is known to the computer, cell position on the slide is a measurement feature of LSCM but cell position cannot be a feature of FCM.
In LSCM, interactions of each cell and the laser are measured and recorded many times in a two dimensional pattern and features computed from these inter-actions are derived. In contrast, in FCM, properties of a single analog pulse are recorded as each cell flows past the laser focus.
With LSCM, because cells are prepared and measured on a slide, it is not necessary to provide single cell or nuclei only cell suspensions. Touch or needle biopsy specimens can be made as imprints or smears or tissue can be measured directly. Cytoplasmic as well as nuclear constituents can be characterized and centrifugations are not required and fewer cells may be lost. Preparations requiring amplification or specific fixatives can be employed without agglutination or cell clumping. The complete area encompassing a specimen is able to be scanned to allow all cells in a small specimen to be measured.
Since the absolute position on the slide of each measured cell's coordinates are recorded in the cell's list of features, the position feature can be used to relocate cells for visual observation or CCD camera image capture. Images may be included in reports or used for high resolution analysis of selected cells. Additionally, cells may be observed and categorized and their category used as values of a category feature for subsequent data analysis. Conversely, cells may be visually located and features of observed cells displayed. The position feature can be treated and displayed as any other feature and used for quality control of staining by displaying a fluorescence feature versus X or Y position.
The LSC.TM. laser scanning cytometer, available from CompuCyte Corporation, makes measurements on each cell at 0.5 micron spatial intervals. Features can be computed such as area, perimeter, the peak value found in the array, and texture, all of which give additional information useful in characterizing cells with fewer dyes and sensors. Constituents that are localized to regions of the cell such as probe spots in fluorescence in situ hybridization (FISH) preparations can be independently characterized yielding other features not obtainable with FCM. The total fluorescence, area, and peak fluorescence of the individual probe spots are used as laser scanning cytometer features allowing the laser scanning cytometer to more accurately count probe spots in cells of FISH specimens.
Cell contouring, i.e., outlining of the cell, is effected by presetting sensors to measure individual position pixel fluorescence values within the cell and to locate pixel positions within the cell where there is a predetermined drop-off of fluorescence value (threshhold value). A position having the predetermined drop-off is a boundary position, with the sum of such sites working to contour the cell.
A list of feature values is computed and stored in a PC computer disk data file for each cell found by the laserscanning cytometer. This list contains the following feature values:
For the sensor used for contouring:
1) The integrated value (the corrected sum of the pixel values in the data contour) equivalent to the FCM constituent value, PA1 2) The peak-value within the data contour, PA1 3) The area of the thresholding contour, PA1 4) The perimeter of the thresholding contour, PA1 5) The absolute slide position of the event's peak value, PA1 6) The computer clock time when the event was measured, PA1 7) The number of probe spots within the cell's data contour for FISH specimens, PA1 8) An annotation feature which the user adds as cells are relocated and visually observed, PA1 9) The structure of the data within the contour is analyzed to determine if the event represents a single or a multiple cell. Multiple cell events are tagged. PA1 1) The integrated value, PA1 2) The peak value within the data contour. PA1 1) The integrated value of all pixels in the probe contour, PA1 2) The area of the probe contour, PA1 3) The distance to the nearest probe spot. PA1 a) utilizing scanning means to examine the cell sample with initial examination parameters, while determining and recording the position of individual cells relative to the scanning means; PA1 b) utilizing the scanning means to examine the cell sample for each of the remainder of the multiple times and determining and recording the position of the individual cells relative to the scanning means during each of said remainder of the multiple times; PA1 c) using the recorded cell position of the individual cells from each of multiple examinations as a key to merge results obtained for individual cells having positions within predetermined deviation values. It is preferred, though not necessary, that the key also includes positions outside of predetermined overlapping distance values from adjacent cells.
For every other sensor:
For every probe spot during FISH applications:
As each specimen is run, the PC computer monitor screen shows a series of windows. Any number of windows containing scatter diagrams of any two features, or histograms of one feature can be displayed. These scattergrams or histograms can be related to the gating region of any other scattergrams or histograms so as to only display cells within the parent display's gating region. Any number of gating regions can be drawn using a mouse. In this way complex relationships involving any sets of features can be developed and used to display subsequent scattergrams or histograms compute a variety of statistics of cells within a region, including sub-population counts and distribution statistics, or to select events for relocation and visualization.
In both FCM and LSCM, it is desirable to simultaneously measure as many constitutents of individual cells as possible. For example, it is desirable to measure the total DNA of each cell simultaneously with two specific DNA sequences in which two fluorescence in situ hybridization probes are each bound to different fluorescent molecules or to simultaneously characterize DNA per cell, cell proliferation, and cell apoptosis. The number of constitutents that can be measured is limited since each laser used emits a single wavelength and can excite dyes to fluoresce only at wavelengths longer in wavelength than the excitation wavelength. Additionally, the excitation bands of many dyes are broad and have grossly different emission intensities and do not allow distinguishing multiple constituents, each stained with different constituent specific dyes. For example, the dye propidium iodide (PI), used to stain DNA, is used in LSCM for both finding and associating each cell's fluoresence digital data for analysis, as well as determining total DNA values per cell. PI is excited by an Argon ion laser and has a broad spectral distribution which does not allow any other dye excited by an Argon ion laser and emitting fluoresence at longer wavelengths than PI to be measured simultaneously with PI, limiting the number of cell constituents that can be measured. Another laser other than the Argon ion laser such as a red light emitting HeNe laser can be used to excite the fluoresence of longer wavelength emitting dyes such as CY3 and CY5 that can be conjugated to antibodies that will bind to specific DNA sequences or specific cell proteins. However, both lasers can not be used simultaneously in LSCM because the direct red light from the HeNe laser will interfere with the measurement of the Argon ion excited dyes since its wavelength is close the Argon ion excited dyes emission wavelength, and the Argon ion excited dyes emission will overlap the emission of the red laser excited dyes. It has therefore not been possible to distinguish more than one additional constituent when using dyes such as PI in a single LSCM assay.